Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Deuterium NMR spectra for the phenyl ring deuterons have been obtained for D-phenylalanine, L-phenylalanine, phenylacetic acid, and phenyl propionic acid in randomly oriented crystals of carboxypeptidase A as a function of water content. The spectra are analyzed using a two-site jump model for phenyl ring pi-flips when the ligand is bound to the protein, and the model includes the possibility that the ligand may exchange with isotropic or unbound environments within the crystal. Although the binding pocket may impose local dynamical constraints, a complete pi-flip motion is consistent with the spectra of all ligands at all water contents. The rate constants for the pi-flip at 298 K are found to be 7.5 x 10(5) S-1, 1.9 x 10(6) S-1, 4.0 x 10(6) S-1, and 4.0 x 10(6) S-1 for L-phenylalanine, D-phenylalanine, phenyl propionic acid, and phenylacetic acid, respectively, at water activity of 0.98. The pi-flip rate for the ligand bound to the enzyme increases with water content. Assuming that the activation barrier may be written, delta G+2 = delta G+2o + baw, where aw is the water activity, and the value of b is -1.9 kcal/mol for phenylacetic acid and phenyl propionic acid, -1.3 kcal/mol for L-phenylalanine, and -2.1 kcal/mol for D-phenylalanine. Phenylacetic acid crystals were studied as an example of a phenyl ring motion that is highly constrained by a known and symmetrical packing environment. The deuterium spectra are complex and are not consistent with pi-flip motions, but they are consistent with a superposition of ring jump motions of 24 degrees, 34 degrees, and 72 degrees, with probabilities in the ratio of 1:1:2. Because of the limited space for motion imposed by the tight packing in the crystal, these motions must be highly cooperative and probably locally coherent; however, the spectra by themselves do not prove this intuitively reasonable hypothesis.
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PMID:Bound ligand motion in crystalline carboxypeptidase A. 899 21

The x-ray crystal structure of recombinant leech-derived tryptase inhibitor (rLDTI) has been solved to a resolution of 1.9 A in complex with porcine trypsin. The nonclassical Kazal-type inhibitor exhibits the same overall architecture as that observed in solution and in rhodniin. The complex reveals structural aspects of the mast cell proteinase tryptase. The conformation of the binding region of rLDTI suggests that tryptase has a restricted active site cleft. The basic amino terminus of rLDTI, apparently flexible from previous NMR measurements, approaches the 148-loop of trypsin. This loop has an acidic equivalent in tryptase, suggesting that the basic amino terminus could make favorable electrostatic interactions with the tryptase molecule. A series of rLDTI variants constructed to probe this hypothesis confirmed that the amino-terminal Lys-Lys sequence plays a role in inhibition of human lung tryptase but not of trypsin or chymotrypsin. The location of such an acidic surface patch is in accordance with the known low molecular weight inhibitors of tryptase.
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PMID:The three-dimensional structure of recombinant leech-derived tryptase inhibitor in complex with trypsin. Implications for the structure of human mast cell tryptase and its inhibition. 924 60

Anahaenopeptins G (1) and H (2) were isolated from the cultured cyanobacterium Oscillatoria agardhii (NIES-595) as potent carboxypeptidase A (CPA) inhibitors. The gross structure of 1 and 2 were established by spectroscopic analysis including the 2D NMR technique and the absolute configurations of 1 and 2 were determined by the spectral and chemical methods. 1 and 2 inhibited CPA with IC50's of 0.0018 and 3.4 microg/mL, respectively.
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PMID:Anabaenopeptins G and H, potent carboxypeptidase A inhibitors from the cyanobacterium Oscillatoria agardhii (NIES-595). 1034 Jun 7

Oligomers of beta-amino acids (beta-peptides), which are readily available by standard meth ods either in solution or on solid support, adopt a large variety of different secondary structures in solution and in the solid state. beta-Peptides 4, 5 and 10 fold into a helix with 3 residues per turn and 14-membered H-bonded rings (314 helix) that is left-handed for 5 and 10 and right-handed for 2 (due to the reversal of the chirality of the building blocks), as was clearly demonstrated by two-dimensional NMR-spectroscopy. This helix thermally is very stable in methanol solution upon heating. As shown by NMR- and CD-spectroscopy, it is partially populated even at 100 C (Figure 3). Another helix was dis covered for mixed beta-peptide 8 in methanol solution: it is characterized by 12- and 10- membered turns (Figure 4, left) and its central 10-membered turn has been found in the solid state of a geminally disubtituted beta-peptide (Figure 4, right). This central 10-membered turn was used as a scaffold to attach beta-amino acid residues that prefer a linear (non-helical) conformation (beta-peptide 21): a hairpin (pleated sheet-turn-pleated sheet) structure was determined in solution by NMR-spectroscopy (Figure 5). In contrast to this antiparallel pleated-sheet, a parallel pleated sheet was found for a beta-tripeptide in the solid state. For the first time it was possible to observe reversible peptide folding in MD simulations by studying beta-peptides (Figure 6) and to determine folding pathways and intermediates. beta-Peptides are a new class of promising peptidomimetics. They are resistant against the degradation by proteolytic enzymes such as pepsin, elastase, carboxypeptidase A, pro nase or proteasom 20S. A variety of beta-amino acids (27-34) was shown to be non- mutagenic by Ames tests and beta-peptides 47 and 48 reveal large elimination half-lives of 3 h (for 47) and 10 h (for 48) in the serum of rodents (Figure 7). Conjugates of alpha- and beta- peptides are efficient ligands for the HLA*B27 MHC Class I protein, a five fold increase of binding (2.0 microM for 55) compared to a natural peptidic ligand 51 was observed. Furthermore, beta-peptides are able to mimic natural a-peptidic hormones such as somatostatin. The cyclo-beta-tetrapeptide 57 binds to the five human somatostatin receptors in the micromolar range. In addition, several other non-natural oligomers such as beta-peptide nucleic acids (built from 58 and 59), beta-peptoids (60), oligomers of anthranilic acids and beta-sulfonamido peptides are discussed.
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PMID:Beta-peptides: twisting and turning. 1051 5

To elucidate intrinsic recognition of carboxamides by zinc(II) in carbonic anhydrase (CA) (as inhibitors) and carboxypeptidase A (CPA) (as substrates), a new series of Zn(2+)-carboxamide-appended cyclen complexes have been synthesized and characterized (cyclen = 1,4,7,10-tetraazacyclododecane). Two types of Zn(2+)-carboxamide interactions have been found. In the first case represented by a zinc(II) complex of carbamoylmethyl-1,4,7,10-tetraazacyclododecane (L(1)), the amide oxygen binds to zinc(II) at slightly acidic pH (to form ZnL(1)), and the deprotonated amide N(-) binds to zinc(II) at alkaline pH (to form ZnH(-1)L(1)) with pK(a) = 8.59 at 25 degrees C and I = 0.1 (NaNO(3)), as determined by potentiometric pH titrations, infrared spectral changes, and (13)C and (1)H NMR titrations. The X-ray crystal structure of ZnH(-1)L(3) (where L(3) = N-(4-nitrophenyl)carbamoylmethyl cyclen, pK(a) = 7.01 for ZnL(3) <==> ZnH(-1)L(3)) proved that the zinc(II) binds to the amidate N(-) (Zn-N(-) distance of 1.974(3) A) along with the four nitrogen atoms of cyclen (average Zn-N distance 2.136 A). Crystal data: monoclinic, space group P2(1)/n (No. 14) with a = 10.838(1) A, b = 17.210(2) A, c = 12.113(2) A, b = 107.38(1) degrees, V = 2156.2(5) A(3), Z = 4, R = 0.042, and R(w) = 0.038. These model studies provide the first chemical support that carboxamides are CA(-) inhibitors by occupying the active Zn(2+) site both in acidic and alkaline pH to prevent the occurrence of the catalytically active Zn(2+)-OH(-) species. In the second case represented by a zinc(II) complex of 1-(N-acetyl)aminoethylcyclen, ZnL(6), the pendant amide oxygen had little interaction with zinc(II) at acidic pH. At alkaline pH, the monodeprotonation yielded a zinc(II)-bound hydroxide species ZnL(6)(OH(-)) (pK(a) = 7.64) with the amide pendant remaining intact. The ZnL(6)(OH(-)) species showed the same nucleophilic activity as Zn(2+)-cyclen-OH(-). The second case may mimic the Zn(2+)-OH(-) mechanism of CPA, where the nucleophilic Zn(2+)-OH(-) species does not act as a base to deprotonate a proximate amide.
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PMID:Study of pH-dependent zinc(II)-carboxamide interactions by zinc(II)-carboxamide-appended cyclen complexes (cyclen = 1,4,7,10-tetraazacyclododecane). 1205 3

The solution structure and backbone dynamics of the recombinant potato carboxypeptidase inhibitor (PCI) have been characterized by NMR spectroscopy. The structure, determined on the basis of 497 NOE-derived distance constraints, is much better defined than the one reported in a previous NMR study, with an average pairwise backbone root-mean-square deviation of 0.5 A for the well-defined region of the protein, residues 7-37. Many of the side-chains show now well-defined conformations, both in the hydrophobic core and on the surface of the protein. Overall, the solution structure of free PCI is similar to the one that it shows in the crystal of the complex with carboxypeptidase A. However, some local differences are observed in regions 15-21 and 27-29. In solution, the six N-terminal and the two C-terminal residues are rather flexible, as shown by 15N backbone relaxation measurements. The flexibility of the latter segment may have implications in the binding of the inhibitor by the enzyme. All the remaining residues in the protein are essentially rigid (S2 > 0.8) with the exception of two of them at the end of a short 3/10 helix. Despite the small size of the protein, a number of amide protons are protected from exchange with solvent deuterons. The slowest exchanging protons are those in a small two-strand beta-sheet. The unfolding free energies, as calculated from the exchange rates of these protons, are around 5 kcal/mol. Other protected amide protons are located in the segment 7-12, adjacent to the beta-sheet. Although these residues are not in an extended conformation in PCI, the equivalent residues in structurally homologous proteins form a third strand of the central beta-sheet. The amide protons in the 3/10 helix are only marginally protected, indicating that they exchange by a local unfolding mechanism, which is consistent with the increase in flexibility shown by some of its residues. Backbone alignment-based programs for folding recognition, as opposite to disulfide-bond alignments, reveal new proteins of unrelated sequence and function with a similar structure.
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PMID:Structure and dynamics of the potato carboxypeptidase inhibitor by 1H and 15N NMR. 1255 84

Inflammatory peptides display different types of post-transcriptional modifications, such as C-terminal amidation, that alter their biological activity. Here we describe the structural and molecular dynamics features of the mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF-NH(2)), found in the venom of the solitary wasp, and of its carboxyl-free C-terminal form (EMP-AF-COO(-)) characterized by a reduced activity. Circular dichroism indicates that both peptides switch from a random coil conformation in water to a helical structure in TFE and SDS micelles. NMR data, in 30% TFE, reveal that the two peptides fold into an alpha-helix spanning most of their length, while they differ in terms of molecular rigidity. To understand the origins of the conformational flexibility observed in the case of EMP-AF-COO(-), a 5 ns MD simulation was carried out for each peptide, in an explicit water/TFE environment. The results show that the two peptides differ in an H-bond between Leu14 NH(2) and the backbone carbonyl of Ile11. The loss of that H-bond in EMP-AF-COO(-) leads to a significant modification of its structural dynamics. In fact, as evidenced by essential dynamics analysis, while EMP-AF-NH(2) exists mainly as a rigid structure, EMP-AF-COO(-) presents two helical stretches that fluctuate in some sort of independent fashion. We conclude that the diverse biological activity of the two peptides is not simply due to the reduction of the net positive charge, as generally suggested, but also to a structural perturbation of the amphipathic alpha-helix that affects their ability to perturb the cell membrane.
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PMID:How C-terminal carboxyamidation alters the biological activity of peptides from the venom of the eumenine solitary wasp. 1513 35

Resistance to proteolytic enzymes and heat is thought to be a prerequisite property of food allergens. Allergens from peanut (Arachis hypogaea) are the most frequent cause of fatal food allergic reactions. The allergenic 2S albumin Ara h 2 and the homologous minor allergen Ara h 6 were studied at the molecular level with regard to allergenic potency of native and protease-treated allergen. A high-resolution solution structure of the protease-resistant core of Ara h 6 was determined by NMR spectroscopy, and homology modelling was applied to generate an Ara h 2 structure. Ara h 2 appeared to be the more potent allergen, even though the two peanut allergens share substantial cross-reactivity. Both allergens contain cores that are highly resistant to proteolytic digestion and to temperatures of up to 100 degrees C. Even though IgE antibody-binding capacity was reduced by protease treatment, the mediator release from a functional equivalent of a mast cell or basophil, the humanized RBL (rat basophilic leukaemia) cell, demonstrated that this reduction in IgE antibody-binding capacity does not necessarily translate into reduced allergenic potency. Native Ara h 2 and Ara h 6 have virtually identical allergenic potency as compared with the allergens that were treated with digestive enzymes. The folds of the allergenic cores are virtually identical with each other and with the fold of the corresponding regions in the undigested proteins. The extreme immunological stability of the core structures of Ara h 2 and Ara h 6 provides an explanation for the persistence of the allergenic potency even after food processing.
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PMID:Structure and stability of 2S albumin-type peanut allergens: implications for the severity of peanut allergic reactions. 1637

An artificial model for the natural enzyme carboxypeptidase A has been constructed by molecular imprinting in synthetic polymers. The tetrahedral transition state analogues (TSAs 4 and 5) for the carbonate hydrolysis have been designed as templates to allow incorporation of the main catalytic elements, an amidinium group and a Zn(2+) or Cu(2+) center, in a defined orientation in the transition state imprinted active site. The complexation of the functional monomer and the template in presence of Cu(2+) through stoichiometric noncovalent interaction was established on the basis of (1)H NMR studies and potentiometric titration. The Cu(2+) center was introduced into the imprinted cavity during polymerization or by substitution of Zn(2+) in Zn(2+) imprinted polymers. The direct introduction displayed obvious advantages in promoting catalytic efficiency. With substrates exhibiting a very similar structure to the template, an extraordinarily high enhancement of the rate of catalyzed to uncatalyzed reaction (k(cat)/k(uncat)) of 10(5)-fold was observed. If two amidinium moieties are introduced in proximity to one Cu(2+) center in the imprinted cavity by complexation of the functional monomer 3 with the template 5, the imprinted catalysts exhibited even higher activities and efficiencies for the carbonate hydrolysis with k(cat)/k(uncat) as high as 410,000. These are by far the highest values obtained for molecularly imprinted catalysts, and they are also considerably higher compared to catalytic antibodies. Our kinetic studies and competitive inhibition experiments with the TSA template showed a clear indication of a very efficient imprinting procedure. In addition, this demonstrates the important role of the transition state stabilization during the catalysis of this reaction.
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PMID:Functional mimicry of carboxypeptidase A by a combination of transition state stabilization and a defined orientation of catalytic moieties in molecularly imprinted polymers. 1851 Mar 22

Three novel structurally related pentadecapeptides, named lasioglossins, were isolated from the venom of the eusocial bee Lasioglossum laticeps. Their primary sequences were established as H-Val-Asn-Trp-Lys-Lys-Val-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-I), H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-II) and H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Lys-NH(2) (LL-III). These lasioglossins exhibited potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, low haemolytic and mast cell degranulation activity, and a potency to kill various cancer cells in vitro. The lasioglossin CD spectra were measured in the presence of trifluoroethanol and sodium dodecyl sulfate solution and indicated a high degree of alpha-helical conformation. NMR spectroscopy, which was carried out in trifluoroethanol/water confirmed a curved alpha-helical conformation with a concave hydrophobic and convex hydrophilic side. To understand the role of this bend on biological activity, we studied lasioglossin analogues in which the Gly in the centre of the molecule was replaced by other amino acid residues (Ala, Lys, Pro). The importance of the N-terminal part of the molecule to the antimicrobial activity was revealed through truncation of five residues from both the N and C termini of the LL-III peptide. C-terminal deamidation of LL-III resulted in a drop in antimicrobial activity, but esterification of the C terminus had no effect. Molecular modelling of LL-III and the observed NOE contacts indicated the possible formation of a bifurcated H-bond between hydrogen from the Lys15 CONH peptide bond and one H of the C-terminal CONH(2) to the Ile11 oxygen atom. Such interactions cannot form with C-terminal esterification.
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PMID:Lasioglossins: three novel antimicrobial peptides from the venom of the eusocial bee Lasioglossum laticeps (Hymenoptera: Halictidae). 1959 Nov 85


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